2. Academic Validation
  3. Arenobufagin Promoted Oxidative Stress-Associated Mitochondrial Pathway Apoptosis in A549 Non-Small-Cell Lung Cancer Cell Line

Arenobufagin Promoted Oxidative Stress-Associated Mitochondrial Pathway Apoptosis in A549 Non-Small-Cell Lung Cancer Cell Line

  • Evid Based Complement Alternat Med. 2020 Apr 22;2020:8909171. doi: 10.1155/2020/8909171.
Jun Kan 1,  Haifu Huang 2,  Zhangyu Jiang 3,  Ruisheng Zhou 3,  Shasha Bai 3,  Caijie Liao 3,  Jiancong Chen 3,  Jun Dong 4,  Yunlong Zhang 5,  Jingzhi Zhang 6,  Rong Zhang 7,  Daihan Zhou 1,  Enxin Zhang 1 2
Affiliations

Affiliations

  • 1 Department of Oncology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
  • 2 Shenzhen Hospital (Futian) of Guangzhou University of Chinese Medicine, Shenzhen 518000, China.
  • 3 Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
  • 4 Department of Integrated Therapy in Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangzhou 510060, China.
  • 5 Key Laboratory of Neuroscience, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, China.
  • 6 The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 510260, China.
  • 7 Institute of Clinical Pharmacology, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
PMID: 32382311 DOI: 10.1155/2020/8909171
Abstract

Arenobufagin (ARE) has demonstrated potent Anticancer activity in various types of tumor, but the role and mechanism of ARE for lung Cancer remain unclear. Oxidative stress exists under normal conditions and is an inevitable state in the body. A variety of noxious stimuli can break the equilibrium state of oxidative stress and promote Apoptosis. Here, we used a CCK-8 assay to examine cell viability. We determined oxidative stress damage by measuring levels of intracellular ROS and levels of GSH, SOD, and MDA. Annexin V-FITC/PI double staining assay, as well as the Hoechst 33258 staining, was used to detect ARE-induced Apoptosis in A549 cell. Evaluation of the expression level of the specified molecule was indicated by Western blot and qRT-PCR. Loss of function experiment was carried out using NAC pretreatment. The experimental results show that ARE significantly declines in the viability of A549 cells and increases the Apoptosis rate of A549 cells. As reflected in cell morphology, the A549 cells showed features of shrinkage and had incompletely packed membranes; the same phenomenon is manifested in Hoechst 33258 staining. Following ARE treatment, the ROS level in A549 cells was rising in a concentration-dependent manner, and so were MDA and GSH levels, while the SOD level was decreasing. Moreover, we found that ARE can decrease mitochondrial membrane potential (MMP), and a cascade of apoptotic processes can be triggered by decreased MMP. Importantly, we found significant changes in protein expression levels and mRNA levels of apoptosis-related proteins. Furthermore, when we used NAC to restrain oxidative stress, the expression levels of apoptosis-related proteins have also changed accordingly. Our data demonstrate that Apoptosis in the non-small-cell lung Cancer (NSCLC) cell line A549 is caused by oxidative stress due to ARE. Our research also shows that ARE may have the potential to become a targeted therapeutic for the treatment of NSCLC in the future.

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